Novel CD20 proteins

Abstract

The present invention relates to a human CD20 protein of which one or more intracellular domains are modified sufficient to reduce or eliminate intracellular signaling when attached to or bound to a cell membrane, such as T cells, natural killer cells, B cells, myeloid cells, pluripotent stem cells, and hematopoietic stem cells. The invention also relates to a nucleic acid encoding the modified human CD20 protein described herein, and the use of the modified human CD20 protein as a safety switch in chimeric antigen receptor T cell therapy or as a selection marker in gene therapy.

Description

TECHNICAL FIELD

[0001] The present invention relates to a human CD20 protein comprising at least one mutation intended to reduce or eliminate intracellular signaling when it is attached to or bound to the cell membrane of a cell, including, for example, T cells, natural killer cells, B cells, myeloid cells, hematopoietic stem cells, non-hematopoietic stem cells, pluripotent stem cells, and human cell lines. In particular, the present invention provides an engineered human CD20 protein comprising at least one mutation in at least one intracellular domain, including amino acid residues 1-56 or 210-297 as defined by SEQ ID NO: 1. BACKGROUND

[0003] The following description contains information that can be useful in understanding the present invention. It is not an admission that any of the information, publications, or documents expressly or implicitly referenced in this section is prior art or necessary to describe or enable the invention described or claimed herein. All publications and patents mentioned herein are incorporated herein by reference in their entirety.

[0004] It is estimated that the U.S. Food and Drug Administration (FDA) will approve 10 to 20 gene therapies per year by 2025 [1]. The rapid development in this field, which targets a variety of diseases including blindness, immune and neuronal diseases, and cancer [2], highlights the urgent need for safety mechanisms to address potential short- and long-term adverse effects, such as cytotoxicity in CAR-T cell therapy.

[0005] Adverse effects arising from gene therapy can be regulated by incorporating safety switches, such as suicide genes, in the gene construct or by including a cell clearance marker that is not normally present in the modified cells, allowing for antibody-mediated cytotoxicity [3]. Using antibodies that have already received clinical approval, such as Cetuximab (anti-EGFR) [4] or Rituximab (anti-CD20) [5], provides an advantage for cell surface clearance markers compared to other types of regulation. The Federal Drug Administration (FDA) and the European Medicines Agency (EMA) recommend evaluation of the cell kinetics, biodistribution, and persistence of gene-modified cells, and long-term follow-up, including monitoring for late-onset events, including secondary malignancies, which can be addressed by using a single protein that serves as both a safety switch and a detection marker [6, 7].

[0006] The present invention aims to address this clinical need by providing an engineered CD20 protein in which the intracellular domain has been modified to eliminate CD20-mediated signaling while retaining detection and killing by anti-CD20 antibodies, including, for example, Rituximab, Obinutuzumab, and Ocrelizumab. SUMMARY

[0007] The invention described and claimed herein has various attributes and embodiments, including but not limited to those set forth, described or referenced in the SUMMARY. The invention is not intended to cover all possibilities, the invention described and claimed herein is not limited to or by the features or embodiments identified in the SUMMARY, and the SUMMARY is intended for illustrative purposes only and not for limiting purposes.

[0008] In one aspect, the invention provides a modified human CD20 protein comprising:

[0009] (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 set forth in SEQ ID NO: 1 ;

[0010] (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 set forth in SEQ ID NO: 1 ;

[0011] (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1 ;

[0012] (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1 ; or

[0013] (v) a combination comprising any one of (i) to (iv)

[0014] wherein the at least one mutation defined by any one of (i) to (v) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0015] In another aspect, the invention provides a modified human CD20 protein comprising:

[0016] (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 set forth in SEQ ID NO: 1 ; and

[0017] (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1,

[0018] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0019] In another aspect, the present application provides a modified human CD20 protein comprising:

[0020] (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 as set forth in SEQ ID NO: 1; and

[0021] (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 as set forth in SEQ ID NO: 1,

[0022] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0023] In another aspect, the present application provides a modified human CD20 protein comprising:

[0024] (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 as set forth in SEQ ID NO: 1; and

[0025] (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 as set forth in SEQ ID NO: 1,

[0026] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0027] In another aspect, the present application provides a modified human CD20 protein comprising:

[0028] (i) truncation of amino acid residues 1-50 as set forth in SEQ ID NO: 1; and

[0029] (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 as set forth in SEQ ID NO: 1,

[0030] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0031] In another aspect, the present application provides a modified human CD20 protein comprising:

[0032] (i) truncation of amino acid residues 1-50 as set forth in SEQ ID NO: 1; and

[0033] (ii) at least one mutation of S225, S231, and T239 as set forth in SEQ ID NO: 1

[0034] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0035] In another aspect, the application provides a modified human CD20 protein comprising:

[0036] (i) a truncation of amino acid residues 1-50 as set forth in SEQ ID NO: 1, and

[0037] (ii) at least one mutation of S225, S231, and T239 as set forth in SEQ ID NO: 1, comprising one or more of S225A, S231A, and T239A,

[0038] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0039] In another aspect, the application provides a modified human CD20 protein comprising:

[0040] (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 as set forth in SEQ ID NO: 1; and

[0041] (ii) a truncation of amino acid residues 253-297 as set forth in SEQ ID NO: 1,

[0042] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0043] In another aspect, the application provides a modified human CD20 protein comprising:

[0044] (i) at least one mutation of T2, T3, S7, T11, S35, S36, and T51 as set forth in SEQ ID NO: 1; and

[0045] (ii) a truncation of amino acid residues 253-297 as set forth in SEQ ID NO: 1,

[0046] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0047] In another aspect, the present application provides a modified human CD20 protein comprising:

[0048] (i) at least one mutation of T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1, comprising one or more of T2A, T3A, S7A, T11A, S35A, S36A and T51A; and

[0049] (ii) a truncation of amino acid residues 253-297 of SEQ ID NO: 1,

[0050] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0051] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 2, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 2.

[0052] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 3, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 3.

[0053] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 4, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 4.

[0054] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 5, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 5.

[0055] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 6, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 6.

[0056] In yet another aspect, the present application provides a modified human CD20 protein comprising the sequence set forth in SEQ ID NO: 7, or a variant sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 7.

[0057] In yet another aspect, the present application provides a modified human CD20 protein comprising:

[0058] (i) at least one mutation that results in truncation of any one of amino acid residues 1-56 of SEQ ID NO: 1;

[0059] (ii) at least one mutation that results in truncation of any one of amino acid residues 210-297 of SEQ ID NO: 1;

[0060] (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 of SEQ ID NO: 1;

[0061] (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 of SEQ ID NO: 1; or

[0062] (v) a combination comprising any one of (i) to (iv); and

[0063] (vi) at least one mutation of any one of amino acid residues 142-188 of SEQ ID NO: 1 that abrogates binding to the monoclonal antibody obinutuzumab

[0064] wherein any one of (i) to (v) results in reduced or abrogated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0065] In yet another aspect, the present application provides a modified human CD20 protein comprising:

[0066] (i) at least one mutation that results in truncation of any one of amino acid residues 1-56 of SEQ ID NO: 1 ;

[0067] (ii) at least one mutation that results in truncation of any one of amino acid residues 210-297 of SEQ ID NO: 1 ;

[0068] (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 of SEQ ID NO: 1 ;

[0069] (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 of SEQ ID NO: 1 ; or

[0070] (v) a combination comprising any one of (i) to (iv); and

[0071] (vi) at least one mutation in any one of amino acid residues 142-188 of SEQ ID NO: 1 that abrogates binding to the monoclonal antibody rituximab

[0072] wherein any one of (i) to (v) results in reduced or abrogated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0073] In another aspect, the application provides a cell expressing any of the modified human CD20 proteins as described herein.

[0074] In another aspect, the application provides a T cell expressing any of the modified human CD20 proteins as described herein.

[0075] In another aspect, the application provides a natural killer (NK) cell expressing any of the modified human CD20 proteins as described herein.

[0076] In another aspect, the application provides a B cell expressing any of the modified human CD20 proteins as described herein.

[0077] In another aspect, the application provides a myeloid cell expressing any of the modified human CD20 proteins as described herein.

[0078] In another aspect, the application provides a pluripotent cell expressing any of the modified human CD20 proteins as described herein.

[0079] In another aspect, the application provides a hematopoietic stem cell expressing any of the modified human CD20 proteins as described herein.

[0080] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein.

[0081] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein.

[0082] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein.

[0083] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein.

[0084] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein.

[0085] In another aspect, the present application provides a non-haematopoietic stem cell line expressing any of the modified human CD20 proteins as described herein. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 Flow cytometry plots showing HEK293 cells transfected with CD20-GFP and CD20form-GFP. Raji B cell line was used as a positive control for CD20 membrane detection.

[0087] Figure 2 Predicted phosphorylation sites of CD20 are shown. bold / underlined Residues indicated are amino acids identical to mouse sequence and with a NetPhos score higher than 0.7; residues within (parentheses) are amino acids identified as identical to mouse sequence; residues within [brackets] are amino acids with a NetPhos score higher than 0.7; grey residues are amino acids identified by crystal structure; gray underlined Residues are amino acids identified using all three methods, reference example 2.

[0088] Figure 3 Detection results of surface CD20WT or modified CD20 on HEK293 cell lines (upper panel) and primary T cells (lower panel) are shown by labelling the cells with Rituximab coupled to AF405.

[0089] Figure 4A Rituximab-mediated total cell death of CD20KO HG3 cells, which were either untransduced or transduced with CD20WT, C1C3 or C1C 252 Transduction.

[0090] Figure 4Bshows the transduction efficiency of CD20WT, C1C3 or C1C 252

[0091] Figure 5 shows the remaining GFP+ cells in the blood of mice (percentage (%) of CD45.1 cells) after treatment with 2H7 antibody or isotype control. Bar graphs represent the mean percentage (%) of 5 mice, error bars represent SEM.

[0092] Figure 6 shows the detection of cell surface CD20 by obinutuzumab, rituximab or L27 antibody in CD20KO HG3 cells transduced with CD20WT, C1C3 or C1C 252

[0093] Figure 7 shows the induction of Ca 252 2+

[0094] Figure 8A shows the global phosphorylation status of CREB, WINK, GSK, ERK, STAT 5 and 6, Lyn, P53 and RSK after activation of CD20 by rituximab binding as measured by Phospho-Western Blot.

[0095] Figure 8B shows the differences in phosphorylation of kinases associated with B cell and T cell activation. Bar graphs represent the mean difference, dots represent individual measurements of 2 replicates per condition.

[0096] Figure 9A shows the identification of key binding residues in the CD20 epitope required for rituximab and obinutuzumab based on published information [8]. The asparagine (N) residue was individually substituted by alanine (A) to specifically eliminate binding of one or the other antibody.

[0097] Figure 9B shows the detection of surface CD20 by rituximab or obinutuzumab in HEK293 cells transduced with CD20WT, Δobinutuzumab mutant (N176A substitution to specifically eliminate obinutuzumab binding) and Δrituximab mutant (N171A substitution to specifically eliminate rituximab binding).

[0098] Figure 10 ​​​​The amino acid sequence of an exemplary modified human CD20 protein according to the present application is shown. X = any naturally or non-naturally occurring amino acid residue. DETAILED DESCRIPTION

[0099] GENERAL DEFINITIONS

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, such as immunology, immunohistochemistry, protein chemistry and biochemistry.

[0101] Unless otherwise indicated, the recombinant protein and immunological techniques used in the present application are standard procedures, well known to those skilled in the art. These techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988); and J.E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0102] The term "and / or", e.g., "A and / or B" shall be understood to mean either "A and B" or "A or B" and shall be taken to provide explicit support for both alternatives (or for either alternative).

[0103] The term "a" or "an" refers to one or more than one of the specified entity; for example, "a subject" or "a nucleic acid molecule" can refer to one or more subjects or nucleic acid molecules, or at least one subject or nucleic acid molecule. Accordingly, the terms "a" or "an", "one or more" and "at least one" can be used interchangeably herein.

[0104] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one or more (i.e. one or a plurality) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0105] References to ranges of numbers (for example 1 to 10) disclosed herein are also to be taken to include references to all individual numbers within that range (for example 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and to any rational number range within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and thus all sub-ranges of all ranges explicitly disclosed herein are hereby expressly disclosed. These are merely specific examples and all possible combinations of the minimum and maximum values within any given range are to be taken as being explicitly stated in a similar manner.

[0106] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0107] Those skilled in the art will appreciate that the subject matter described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the application includes all such variations and modifications. The application also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features.

[0108] The application is not limited to the particular embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the application, as described herein.

[0109] Any embodiment or implementation described herein should be considered to apply mutatis mutandis to any other embodiment or implementation, unless specifically stated otherwise.

[0110] Selected Definitions

[0111] The term "amino acid residue" or "amino acid" refers to an amino acid incorporated into a protein, polypeptide, or peptide. The term "polypeptide" includes any polymer of amino acids or amino acid residues. The term "polypeptide sequence" refers to a series of amino acids or amino acid residues that physically make up a polypeptide. A "protein" is a macromolecule comprising one or more polypeptides or polypeptide "chains." A "peptide" is a small polypeptide, usually less than 15-20 amino acid residues in total. The term "amino acid sequence" refers to a series of amino acids or amino acid residues that physically make up a peptide or polypeptide, depending on its length. Unless otherwise indicated, the polypeptide and protein sequences disclosed herein are written left to right in the order of amino to carboxy terminus.

[0112] The terms "amino acid," "amino acid residue," "amino acid sequence," or polypeptide sequence include the naturally occurring amino acids, including both the L- and D- isomers, unless otherwise indicated, and also known natural amino acid analogs that function in a similar manner to naturally occurring amino acids, such as selenocysteine, pyrrolysine, N- formylmethionine, gamma-carboxyglutamate, hydroxyproline, pyridoxine, and selenomethionine. Amino acids referred to herein are described in shorthand form as shown in Table A:

[0113] Table A: Amino Acid Nomenclature

[0114] Name 3-letter code 1 -letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp T Tyrosine Tyr Y Valine Val V

[0115] The term "antibody" refers to an immunoglobulin molecule capable of selective binding to a target (e.g., human CD20) by means of an antigen binding site contained within at least one variable region. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatised antibodies, de-immunized antibodies, half-antibodies, bispecific antibodies), and single domain antibodies such as domain antibodies and heavy chain only antibodies (e.g., camelid antibodies or cartilaginous fish immunoglobulin new antigen receptor (IgNAR)). Antibodies typically comprise constant domains which can be arranged into constant regions or constant fragments or fragment crystallizable (Fc). Preferred forms of antibodies comprise a four-chain structure as their basic unit. Full length antibodies comprise two covalently linked heavy chains (-50-70 kDa) and two light chains (-23 kDa each). Light chains generally comprise a variable region and a constant domain, with light chains being either kappa light chains or lambda light chains in mammals. Heavy chains generally comprise a variable region and one or two constant domains connected by a hinge region to other constant domains. Heavy chains in mammals belong to one of five types: alpha, delta, epsilon, gamma, or mu. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains and heavy and light chains are held together by interchain disulfide bonds and noncovalent interactions. The number of interchain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each of which is -110 amino acids in length) and one or more constant regions at the C-terminus. The constant region of the light chain (CL, -110 amino acids in length) aligns with the first constant region of the heavy chain (CH, -330-440 amino acids in length) and is connected by a disulfide bond. The light chain variable region aligns with the variable region of the heavy chain. Antibody heavy chains can comprise 2 or more additional CH domains (e.g., CH2, CH3, etc.) and can comprise a hinge region identified between the CH1 and Cm constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (preferably human) antibody. The term "antibody" encompasses not only intact polyclonal antibodies or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion with an antigen binding site, humanized antibodies, human antibodies, chimeric antibodies, primatised antibodies, de-immunized antibodies, or veneered antibodies.

[0116] For polypeptides, the term "conservative substitution" refers to changes in the amino acid composition of a polypeptide that do not significantly alter the function and structure of the overall polypeptide (see Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, New York (2d ed. 1992)).

[0117] As used herein, the term "expression" and grammatical variants thereof refer to the transcription and / or translation of a polynucleotide or nucleic acid into a polypeptide or protein. The resulting polypeptide or protein can remain within a cell, become a component of a cell surface membrane, or be secreted into the extracellular space.

[0118] As used herein, a cell that expresses a substantial amount of CD20 on at least one cell surface is a "CD20-positive cell" or "CD20 + cell", and is a cell that physically couples to a substantial amount of the extracellular target biomolecule CD20.

[0119] The term "encode", as used herein, refers to the inherent property of specific sequences of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as templates for synthesizing other sequences of nucleotides (e.g., rRNA, tRNA, and mRNA) or of amino acids, and the biological properties that result therefrom. Thus, if a protein is produced in a cell or other biological system in response to transcription and translation of mRNA corresponding to a gene, that gene, cDNA, or RNA is said to encode the protein. Both the coding strand, which is used as the template for the mRNA transcript, and the non-coding strand, which has the same sequence as the mRNA except that the nucleotides are the complement of the mRNA, can be referred to as encoding the protein or other product of the gene or cDNA.

[0120] As used herein, the term "epitope" refers to a portion of an antigen (e.g., human CD20) with which an antibody molecule specifically interacts. These portions, referred to herein as epitope determinants, often comprise or are part of elements such as amino acid side chains or sugar side chains. Epitope determinants can be defined by, e.g., methods known in the art or disclosed herein (e.g., by crystallography or hydrogen-deuterium exchange). At least one or some of the portions of an antibody molecule that specifically interact with an epitope determinant are often located in the CDRs. Typically, an epitope has specific three-dimensional structural features. Typically, an epitope has specific charge distribution features. Some epitopes are linear epitopes, while others are conformational epitopes. An exemplary epitope according to the present application is the epitope defined by amino acid residues 167 to 183 of SEQ ID NO: 1, i.e., CEPANPSEKNSPSTQYC (SEQ ID NO: 13), which comprises amino acid residues critical for binding of the clinically approved monoclonal antibodies rituximab and obinutuzumab.

[0121] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of the defined sequences of nucleotides, i.e., rRNA, tRNA, and mRNA, or of other macromolecules such as proteins and have the biological properties that are conferred by them. Thus, if a protein is produced in a cell or other biological system in response to transcription and translation of mRNA, that mRNA is said to encode the protein. The coding strand of a gene or cDNA (which has the same sequence of nucleotides as mRNA, and is usually provided in sequence listings) and the non-coding strand (which has the sequence of nucleotides complementary to mRNA) can both be referred to as encoding the protein or other product of the gene or cDNA.

[0122] As used herein, the term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

[0123] As used herein, the term "exogenous" refers to any substance that is introduced or produced from outside an organism, cell, tissue, or system.

[0124] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0125] The term "expression vector" refers to a vector that comprises a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for use in expression can be provided by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) into which a recombinant polynucleotide is integrated.

[0126] The term "identity" refers to the sequence identity of subunits between two polymeric molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit site in one of the two molecules is occupied by the same monomeric subunit; e.g., if a certain position in each of two polypeptide or protein molecules is occupied by serine, then they are homologous or identical at that position. The homology between two sequences is directly related to the number of matching or homologous positions; e.g., if half of the positions (e.g., 5 positions in a polymer 10 subunits long) in two sequences are identical, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, then the two sequences are 90% identical.

[0127] The term "intracellular signaling domain" as used herein refers to the intracellular portion of a molecule, e.g., CD20. The intracellular signaling domain generates a signal that promotes an immune effector function, e.g., of a cell containing a CAR, e.g., a CAR-T cell. Examples of immune effector functions, e.g., in a CAR-T cell, include cytolysis and helper activities, including secretion of cytokines. In certain examples, the intracellular signaling domain is part of a protein that transmits an effector function signal and directs the cell to perform a particular function. While a complete intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. In terms of using truncated portions of an intracellular signaling domain, they can be used in place of the entire chain so long as such truncated portions transduce an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0128] The term "isolated" as used herein in reference to a protein or polypeptide sequence disclosed herein refers to a sequence that is removed from its natural cell or other naturally occurring biological environment. An isolated molecule can be obtained by any method or combination of methods, including biochemical techniques, recombinant techniques, and synthetic techniques. A polypeptide sequence can be prepared by at least one purification step.

[0129] The term “lentivirus” refers to a species of the family of retroviruses. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of a host cell and are therefore among the most efficient gene delivery vehicles. HIV, SIV, and FIV are examples of lentiviruses.

[0130] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentiviral genome, including specifically self-inactivating lentiviral vectors, such as the vectors provided in

[44] . Other examples of lentiviral vectors that are available for clinical use include, but are not limited to, for example, vectors from Oxford BioMedica Gene Delivery Technologies or Lentigen Vector systems. Non-clinical variants of lentiviral vectors are also available and are known to those skilled in the art.

[0131] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. The term “nucleic acid” includes genes, cDNA, or mRNA. In one example, a nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless otherwise indicated, the term includes nucleic acids containing natural nucleotides analogs or derivatives that have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the

[0132] The term “nucleic acid encoding an amino acid sequence” or “nucleic acid encoding a protein” includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence or protein. In certain examples, a nucleic acid encoding a polypeptide or protein can contain one or more introns.

[0133] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a compound composed of amino acid residues connected by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no maximum limit to the number of amino acids that make up a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids connected by peptide bonds. As used herein, the term refers to both short chains (which are also referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and long chains (which are also referred to in the art as proteins, which have numerous types including, but not limited to, monomeric, dimeric, trimeric, tetrameric, and higher order multimers). "Polypeptide" includes, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0134] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, in the present specification, the terms "nucleic acid" and "polynucleotide" are used interchangeably as the terms are understood by those of ordinary skill in the art in light of the present disclosure. As is understood by those of ordinary skill in the art in light of the present disclosure, a nucleic acid is a polynucleotide, which can be hydrolyzed to its monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, a polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any available method in the art, including, but not limited to, recombinant methods (i.e., cloning nucleic acid sequences from a recombinant library or a cell genome using conventional cloning techniques and PCR, etc.) and by synthetic methods.

[0135] The term "promoter" refers to a DNA sequence recognized by the transcription machinery of the cell or introduced into the cell, which promotes transcription of a particular nucleotide sequence.

[0136] The term "promoter / regulatory sequence" refers to a nucleic acid sequence which is operably linked to a gene to direct expression of the gene product. In some instances, the sequence can be a core promoter sequence; in other instances, the sequence can also include enhancer sequences and other regulatory elements required for expression of the gene product. For example, the promoter / regulatory sequence can be one which expresses the gene product in a tissue-specific manner.

[0137] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a stimulatory tumor antigen) present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample.

[0138] In the present specification, reference to "at least one mutation to a serine or threonine" can include reference to any one or more of T2, T3, S7, T11, S25, S35, S36, T41, S43, S49, T51, S221, S225, S231, T239, T250, T252, S253, S254, T275, T277, S288, S289, S295 and S296 of SEQ ID NO: 1, and includes substitution mutations, deletion mutations or insertion mutations involving amino acid residues equivalent to: threonine at position 2, threonine at position 3, serine at position 7, threonine at position 11, serine at position 25, serine at position 35, serine at position 36, threonine at position 41, serine at position 43, serine at position 49, threonine at position 51, serine at position 221, serine at position 225, serine at position 231, threonine at position 239, threonine at position 250, threonine at position 252, serine at position 253, serine at position 254, threonine at position 275, threonine at position 277, serine at position 288, serine at position 289, serine at position 295 and serine at position 296 as defined by the reference human CD20 sequence set forth in SEQ ID NO: 1.

[0139] The term "variant" as used herein refers to a protein or polypeptide sequence that differs from a specifically identified sequence by, for example, the deletion, substitution, or addition of one or more amino acid residues. Variants can be naturally occurring allelic variants, or non-naturally occurring variants. Variants can be from the same species or from other species and can encompass homologs, paralogs, and orthologs. In certain embodiments, polypeptide variants useful in the present application have the same or similar biological activity as the parent polypeptide, including signal peptide activity or antigen binding properties. The term "variant" with respect to a polypeptide encompasses all forms of polypeptides as defined herein.

[0140] Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the application. Identity can be found over a comparison window of at least 297 amino acid positions for polypeptides.

[0141] Polypeptide variants also encompass those variants that exhibit similarity to one or more specifically identified sequences, which can retain functional equivalence to those sequences, including those variants that cannot reasonably be expected to occur by random chance.

[0142] Identity and similarity of polypeptide sequences can be determined as follows. The BLASTp (BLAST suite of programs, version 2.2.18 [April 2008]) in bl2seq is used to compare a polypeptide sequence of interest to a candidate polypeptide sequence, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are used, but the filtering function for low complexity regions is turned off.

[0143] Similarity of polypeptide sequences can be checked using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp. The parameter -F F turns off filtering of low complexity portions. The parameter -p selects the appropriate algorithm for the pair of sequences. The program looks for regions of similarity between the sequences and reports for each such region of similarity an "E-value" which indicates the number of times such a match would be expected to occur by chance in a database of a fixed reference size containing random sequences. For smaller E-values, far less than 1, this approximates the probability of such a random match. Variant polypeptide sequences typically have an E-value of less than 1 x 10 -5 -6 -9 -12 -15 -18 -21 ​​​​​​The entire length of the overlap between a candidate polypeptide sequence and a polypeptide sequence of interest can also be calculated using a global sequence alignment program. EMBOSS-needle (available at http: / / www.ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235), discussed above, are also global sequence alignment programs suitable for calculating polypeptide sequence identity. According to the present application, BLASTp is preferably used to determine polypeptide variants.

[0144] The term "vector" as used herein refers to the composition of matter comprising an isolated nucleic acid and useful for delivering the isolated nucleic acid to a cell. A variety of vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term "vector" is also understood to include non-plasmid and non-viral compounds that aid in the transfer of nucleic acids into a cell, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0145] The acronym "WT" or "W-T" as used herein is intended to mean "wild type", when used in the context of, for example, human CD20, is intended to mean the composition of the CD20 protein normally found in situ.

[0146] DETAILED DESCRIPTION

[0147] Mutant (human) CD20 - cytoplasmic domain modifications

[0148] The physiological role, regulation and ligand of CD20 are not known [9], but functional studies suggest that CD20 is required for efficient B-cell receptor signalling

[10] and is directly involved in calcium entry, the normal function of which depends on association with lipid rafts

[11] .

[0149] CD20 is the target of clinically approved monoclonal antibodies, including rituximab and obinutuzumab, which are commonly used to deplete lymphocytes in the treatment of B-cell cancers and autoimmune diseases, and have a well-established safety profile.

[0150] If significant on-target off-tissue toxicity or off-target toxicity occurs, it can be necessary to rapidly clear the gene transduced or gene transfected cells. However, the binding of rituximab or obinutuzumab can cause transient activation of CD20 expressing cells by calcium influx and initiation of intracellular signal transduction cascades (e.g., kinase phosphorylation)

[12] ,

[60] ,

[61] . This can result in paradoxical exacerbation of toxicity by the genetically modified cells, especially at risk in recipients with defects in complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) due to recent use of cytotoxic drugs or immunosuppressive agents, or in patients with underlying immunosuppressive conditions.

[0151] The primary mechanisms of cell killing mediated by anti-CD20 antibodies are CDC and ADCC

[13] , although one proposed mechanism of therapeutic B cell apoptosis is direct cytotoxicity mediated by Src family kinases through lipid raft aggregation

[14] . Given that both the N-terminus and C-terminus are intracellular (i.e., as cytoplasmic domains), the applicants designed a series of CD20 truncations and chimeric proteins in an attempt to abrogate CD20 signaling but retain antibody binding, maintaining antibody-dependent cellular apoptosis.

[0152] The initial strategy employed was to truncate the majority of the cytoplasmic domain and combine the cytoplasmic and transmembrane domains of CD20. Significant modifications to the transmembrane 4a (MS4a) protein can affect the protein structure and its ability to traffic to and properly associate with the cell membrane.

[0153] The applicants also combined the minimal antibody binding epitope of CD20 with different signal peptides, transmembrane domains, and cytoplasmic domains known to facilitate good surface protein trafficking and expression on the cell membrane

[15] . The applicants initially tested the human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) signal peptide and the epidermal growth factor receptor (EGFR) transmembrane domain because truncation of the intracellular domain of EGFR results in a membrane-bound protein

[15] .

[0154] Next, the applicants combined different lengths of the antibody binding region of CD20 with the CD28 signal peptide and with the transmembrane and cytoplasmic domains derived from CD28 and contactin-associated protein-like 2 (CASPR2) to generate chimeric proteins. This was because constructs integrating the CD28 and CASPR2 transmembrane / cytoplasmic domains can result in membrane-bound proteins

[16] ,

[17] .

[0155] The different sequence constructs developed for these initial experiments are shown in Table 1 in Example 2.

[0156] The preliminary results (as Figure 1The results were not as expected (see Figure 1). None of the constructs listed in Table 1 were detected on the cell membrane of the HEK293 cell line. However, it is noteworthy that expression of the CD20-encoding transgene was detected in these cells, as reflected in the production of green fluorescent protein tag.

[0157] By way of example only, neither the CD20t v4 construct nor the CD20t v5.3 construct, in which amino acids from the CD20 extracellular domain were fused to different signal peptides (e.g. GM-CSFR and CD28) and transmembrane / cytoplasmic domains (e.g. EGFR and CASPR2), resulted in membrane expression in the HEK293 cell line. This result is particularly surprising in light of previous literature reports documenting that proteins incorporating these domains are able to successfully traffic across the membrane for expression [4, 16, 17].

[0158] In light of these unexpected findings, the Applicant modified its approach to developing a membrane-bound, non-signalling CD20 molecule. See the alternative strategies outlined in Examples 3-6, and read in conjunction with Figures 2-5 .

[0159] Briefly, the Applicant's alternative approach focused on targeting a phosphorylation residue within the cytoplasmic domain of CD20, as CD20 is highly phosphorylated on serine and threonine residues in normal and malignant B cells, and this process is associated with B cell proliferation

[18] . Binding of rituximab to CD20 initiates a signalling cascade that can play a role in antibody-mediated cytotoxicity. CD20 associates with lyn, fyn, lck and p75 / 85 kinases

[19] , which engage to activate PLCy through src family kinases

[20] .

[0160] The cytoplasmic sequence of CD20 contains 15 serine residues, 11 threonine residues, and no tyrosine residues. See Figure 2 . Thus, CD20 has 26 potential phosphorylation sites, although direct evidence for only two sites has been reported in the literature

[21] .

[0161] Accordingly, in one aspect of the application, there is provided a modified human CD20 protein comprising:

[0162] (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 as set out in SEQ ID NO: 1 ;

[0163] (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 as set out in SEQ ID NO: 1 ;

[0164] (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 of SEQ ID NO: 1 ;

[0165] (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 of SEQ ID NO: 1 ; or

[0166] (v) a combination comprising any one of (i) to (iv)

[0167] wherein any one of (i) to (v) results in reduced or eliminated intracellular signaling when the modified CD20 protein is attached to or associated with a cell membrane.

[0168] In one example according to this aspect and all other aspects of the application, the at least one mutation associated with feature (i) results in a truncation of any one of the following amino acid residues set forth in SEQ ID NO: 1 : amino acid residue 1 defined by 5'-M-3' (SEQ ID NO: 26), amino acid residues 1-2 defined by 5'-MT-3' (SEQ ID NO: 27), amino acid residues 1-3 defined by 5'-MTT-3' (SEQ ID NO: 28), amino acid residues 1-4 defined by 5'-MTTP-3' (SEQ ID NO: 29), amino acid residues 1-5 defined by 5'-MTTPR-3' (SEQ ID NO: 30), amino acid residues 1-6 defined by 5'-MTTPRN-3' (SEQ ID NO: 31), amino acid residues 1-7 defined by 5'-MTTPRNS-3' (SEQ ID NO: 32), amino acid residues 1-8 defined by 5'-MTTPRNSV-3' (SEQ ID NO: 33), amino acid residues 1-9 defined by 5'-MTTPRNSVN-3' (SEQ ID NO: 34), amino acid residues 1-10 defined by 5'-MTTPRNSVNG-3' (SEQ ID NO: 35), amino acid residues 1-11 defined by 5'-MTTPRNSVNGT-3' (SEQ ID NO: 36), amino acid residues 1-12 defined by 5'-MTTPRNSVNGTF-3' (SEQ ID NO: 37), amino acid residues 1-13 defined by 5'-MTTPRNSVNGTFP-3' (SEQ ID NO: 38), amino acid residues 1-14 defined by 5'-MTTPRNSVNGTFPA-3' (SEQ ID NO: 39), amino acid residues 1-15 defined by 5'-MTTPRNSVNGTFPAE-3' (SEQ ID NO: 40), amino acid residues 1-16 defined by 5'-MTTPRNSVNGTFPAEP-3' (SEQ ID NO: 41), amino acid residues 1-17 defined by 5'-MTTPRNSVNGTFPAEPM-3' (SEQ ID NO: 42), amino acid residues 1-18 defined by 5'-MTTPRNSVNGTFPAEPMK-3' (SEQ ID NO: 43), amino acid residues 1-19 defined by 5'-MTTPRNSVNGTFPAEPMKG-3' (SEQ ID NO: 44), amino acid residues 1-20 defined by 5'-MTTPRNSVNGTFPAEPMKGP-3' (SEQ ID NO: 45),amino acid residues 1-12 defined by 5’ -MTTPRNSVNGTFPAEPMKGPI-3’ (SEQ ID NO: 46), amino acid residues 1-22 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIA-3’ (SEQ ID NO: 47), amino acid residues 1-23 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAM-3’ (SEQ ID NO: 48), amino acid residues 1-24 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQ-3’ (SEQ ID NO: 49), amino acid residues 1-25 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQS-3’ (SEQ ID NO: 50), amino acid residues 1-26 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSG-3’ (SEQ ID NO: 51), amino acid residues 1-27 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGP-3’ (SEQ ID NO: 52), amino acid residues 1-28 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPK-3’ (SEQ ID NO: 53), amino acid residues 1-29 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKP-3’ (SEQ ID NO: 54), amino acid residues 1-30 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPL-3’ (SEQ ID NO: 55), amino acid residues 1-31 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLF-3’ (SEQ ID NO: 56), amino acid residues 1-32 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFR-3’ (SEQ ID NO: 57), amino acid residues 1-33 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRR-3’ (SEQ ID NO: 58), amino acid residues 1-34 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRM-3’ (SEQ ID NO: 59), amino acid residues 1-35 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMS-3’ (SEQ ID NO: 60), amino acid residues 1-36 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSS-3’ (SEQ ID NO: 61),amino acid residues 1-37 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSL- 3’ (SEQ ID NO: 62), amino acid residues 1-38 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLV- 3’ (SEQ ID NO: 63), amino acid residues 1-39 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG- 3’ (SEQ ID NO: 64), amino acid residues 1-40 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGP- 3’ (SEQ ID NO: 65), amino acid residues 1-41 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPT- 3’ (SEQ ID NO: 66), amino acid residues 1-42 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQ- 3’ (SEQ ID NO: 67), amino acid residues 1-43 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQS- 3’ (SEQ ID NO: 68), amino acid residues 1-44 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSF- 3’ (SEQ ID NO: 69), amino acid residues 1-45 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFF- 3’ (SEQ ID NO: 70), amino acid residues 1-46 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFM- 3’ (SEQ ID NO: 71), amino acid residues 1-47 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMR- 3’ (SEQ ID NO: 72), amino acid residues 1-48 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRE- 3’ (SEQ ID NO: 73), amino acid residues 1-49 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRER- 3’ (SEQ ID NO: 74), amino acid residues 1-50 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRERL- 3’ (SEQ ID NO: 75), amino acid residues 1-51 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRERLK- 3’ (SEQ ID NO: 76), amino acid residues 1-52 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRERLKQ- 3’ (SEQ ID NO: 77), amino acid residues 1-53 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRERLKQE- 3’ (SEQ ID NO: 78), amino acid residues 1-54 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRERLKQEQ- 3’ (SEQ ID NO: 79),amino acid residues 1-49 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRES-3’ (SEQ ID NO: 74), amino acid residues 1-50 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG PTQSFFMRESK-3’ (SEQ ID NO: 75), amino acid residues 1-51 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKT-3’ (SEQ ID NO: 76), amino acid residues 1-52 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG PTQSFFMRESKTL-3’ (SEQ ID NO: 77), amino acid residues 1-53 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLG-3’ (SEQ ID NO: 78), amino acid residues 1-54 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG PTQSFFMRESKTLGA-3’ (SEQ ID NO: 79), amino acid residues 1-55 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAV-3’ (SEQ ID NO: 80), amino acid residues 1-56 defined by 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQ-3’ (SEQ ID NO: 81),

[0169] In another example according to this and other aspects of the application, the at least one mutation associated with feature (i) results in a truncation of amino acid residues 1-50 as set forth in SEQ ID NO: 1, i.e. 5’ -MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVG PTQSFFMRESK-3’ (SEQ ID NO: 75).

[0170] In another example according to this and other aspects of the application, the at least one mutation associated with feature (i) results in a truncation of any one of the following amino acid residues set forth in SEQ ID NO: 1 : amino acid residue 297 defined by 5’-P-3’ (SEQ ID NO: 82), amino acid residues 296-297 defined by 5’-SP-3’ (SEQ ID NO: 83), amino acid residues 295-297 defined by 5’-SSP-3’ (SEQ ID NO: 84), amino acid residues 294-297 defined by 5’-DSSP-3’ (SEQ ID NO: 85), amino acid residues 293-297 defined by 5’-NDSSP-3’ (SEQ ID NO: 86), amino acid residues 292-297 defined by 5’-ENDSSP-3’ (SEQ ID NO: 87), amino acid residues 291-297 defined by 5’-IENDSSP-3’ (SEQ ID NO: 88), amino acid residues 290-297 defined by 5’-PIENDSSP-3’ (SEQ ID NO: 89), amino acid residues 289-297 defined by 5’-SPIENDSSP-3’ (SEQ ID NO: 90), amino acid residues 288-297 defined by 5’-SSPIENDSSP-3’ (SEQ ID NO: 91), amino acid residues 287-297 defined by 5’-ESSPIENDSSP-3’ (SEQ ID NO: 92), amino acid residues 286-297 defined by 5’-QESSPIENDSSP-3’ (SEQ ID NO: 93), amino acid residues 285-297 defined by 5’-DQESSPIENDSSP-3’ (SEQ ID NO: 94), amino acid residues 284-297 defined by 5’-QDQESSPIENDSSP-3’ (SEQ ID NO: 95), amino acid residues 283-297 defined by 5’-PQDQESSPIENDSSP-3’ (SEQ ID NO: 96), amino acid residues 282-297 defined by 5’-PPQDQESSPIENDSSP-3’ (SEQ ID NO: 97), amino acid residues 281-297 defined by 5’-EPPQDQESSPIENDSSP-3’ (SEQ ID NO: 98), amino acid residues 280-297 defined by 5’-PEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 99), amino acid residues 279-297 defined by 5’-FPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 100),Amino acid residues 278-297 defined by 5'–NFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:101), amino acid residues 277-297 defined by 5'–TNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:102), amino acid residues 276-297 defined by 5'–ETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:103), amino acid residues 275-297 defined by 5'–TETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:104), amino acid residues 274-297 defined by 5'–ETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:105), and amino acid residues 278-297 defined by 5'–EETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:101). Amino acid residues 273-297 defined by 5'–EEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:107), amino acid residues 272-297 defined by 5'–EEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:108), amino acid residues 271-297 defined by 5'–EEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:109), amino acid residues 270-297 defined by 5'–EEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:110), amino acid residues 269-297 defined by 5'–QEEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:106), amino acid residues 272-297 defined by 5'–EEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:108), amino acid residues 271-297 defined by 5'–EEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:109), amino acid residues 269-297 defined by 5'–EEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:110), amino acid residues 273-297 defined by 5'–QEEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:106), amino acid residues 272-297 defined by 5'–QEEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:107), amino acid residues 271-297 defined by 5'–QEEEEEETETNFPEPPQDQESSPIENDSSP–3' (SEQ ID NO:108), amino acid residues 270-297 defined Amino acid residues 268-297 defined by SEQ ID NO:111, amino acid residues 267-297 defined by 5'-IQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO:112), amino acid residues 266-297 defined by 5'-PIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO:113), amino acid residues 265-297 defined by 5'-IPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO:114), and amino acid residues 264-297 defined by 5'-IIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO:115).amino acid residues 260-297 defined by 5’-EDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 119), amino acid residues 259-297 defined by 5’-EEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 120), amino acid residues 258-297 defined by 5’-NEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 121), amino acid residues 257-297 defined by 5’-KNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 122), amino acid residues 256-297 defined by 5’-PKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 123), amino acid residues 255-297 defined by 5’-QPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 124), amino acid residues 254-297 defined by 5’-SQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 125), amino acid residues 253-297 defined by 5’-SSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 126), amino acid residues 252-297 defined by 5’-TSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 127), amino acid residues 251-297 defined by 5’-NTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 128), amino acid residues 250-297 defined by 5’-KNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 129), amino acid residues 249-297 defined by 5’-PKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 130), amino acid residues 248-297 defined by 5’-QPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 131), amino acid residues 247-297 defined by 5’-SQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 132), amino acid residues 246-297 defined by 5’-SSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 133), amino acid residues 245-297 defined by 5’-TSSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 134), amino acid residues 244-297 defined by 5’-NTSSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 135), amino acid residues 243-297 defined by 5’-KNTSSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 136), amino acid residues 242-297 defined by 5’-PKNTSSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 137), amino acid residues 241-297 defined by 5’-QPKNTSSQPKNTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3’ (SEQ ID NO: 138),amino acid residues 241-297 defined by 5'-EIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 138), amino acid residues 240-297 defined by 5'-DKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 139), amino acid residues 239-297 defined by 5'-ADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 140), amino acid residues 238-297 defined by 5'-VADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 141), amino acid residues 237-297 defined by 5'-EVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 142), amino acid residues 236-297 defined by 5'-VEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 143), amino acid residues 235-297 defined by 5'-IVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 144), amino acid residues 234-297 defined by 5'-DIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 145), amino acid residues 233-297 defined by 5'-ADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 146), amino acid residues 232-297 defined by 5'-VADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 147), amino acid residues 231-297 defined by 5'-EVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 148), amino acid residues 230-297 defined by 5'-VEVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 149), amino acid residues 229-297 defined by 5'-IVEVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 150), amino acid residues 228-297 defined by 5'-DIVEVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 151), amino acid residues 227-297 defined by 5'-ADIVEVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 152), amino acid residues 226-297 defined by 5'-VADIVEVADIVEVADKEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 153), amino acid residues 225-297 defined by 5'-amino acid residues 239-297 defined by 5'-TIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 140), amino acid residues 238-297 defined by 5'-QTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 141), amino acid residues 237-297 defined by 5'-EQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 142), amino acid residues 236-297 defined by 5'-KEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 143), amino acid residues 235-297 defined by 5'-KKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 144), amino acid residues 234-297 defined by 5'-EKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 145), amino acid residues 233-297 defined by 5'-EEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 146), amino acid residues 232-297 defined by 5'-AEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 147), amino acid residues 231-297 defined by 5'-AAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 148), amino acid residues 230-297 defined by 5'-NAAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 149), amino acid residues 229-297 defined by 5'-NNA AEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 150), amino acid residues 228-297 defined by 5'-NNNAAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 151), amino acid residues 227-297 defined by 5'-NNNNNAAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3' (SEQ ID NO: 152), amino acid residues 226-297 defined by 5'-amino acid residues 231-297 defined by 5’ -SAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 148), amino acid residues 230-297 defined by 5’ -LSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 149), amino acid residues 229-297 defined by 5’ -LLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 150), amino acid residues 228-297 defined by 5’ -VLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 151), amino acid residues 227-297 defined by 5’ -IVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 152), amino acid residues 226-297 defined by 5’ -NIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 153), amino acid residues 225-297 defined by 5’ -SNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 154), amino acid residues 224-297 defined by 5’ -KSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 155), amino acid residues 223-297 defined by 5’ -PKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP -3’ (SEQ ID NO: 156),amino acid residues 222-297 defined by 5'-RPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 157), amino acid residues 221-297 defined by 5'-SRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 158), amino acid residues 220-297 defined by 5'-CSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 159), amino acid residues 219-297 defined by 5'-TCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 160), amino acid residues 218-297 defined by 5'-RTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 161), amino acid residues 217-297 defined by 5'-KRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 162), amino acid residues 216-297 defined by 5'-WKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 163), amino acid residues 215-297 defined by 5'-EWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP-3' (SEQ ID NO: 164),amino acid residues 211-297 defined by 5’ - IVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP - 3’ (SEQ ID NO: 168), amino acid residues 210-297 defined by 5’ - GIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP - 3’ (SEQ ID NO: 169).

[0171] In another example according to this and other aspects of the application, the at least one mutation associated with feature (ii) results in a truncation of amino acid residues 253-297 as set forth in SEQ ID NO: 1, i.e., 5’ - SSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP - 3’ (SEQ ID NO: 126).

[0172] In another example according to this and other aspects of the application, the at least one mutation of a threonine or serine located in amino acid residues 1-56 as set forth in SEQ ID NO: 1 comprises a mutation of T2, T3, S7, T11, S25, S35, S36, T41, S43, S49, and T51.

[0173] In yet another example, the mutation of the at least one threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1 comprises a mutation of T83, S221, S225, S231, T239, T250, T252, S253, S254, T275, T277 S288, S289, S295, and S296.

[0174] To reduce the number of potential mutagenesis targets, and thus the number of protein structure alterations, the applicants employed three different predictive tools to select potential phosphorylation modification sites: (i) NetPhos 3.1, which uses a collection of neural networks to predict S, T, or Y phosphorylation sites in eukaryotic proteins; (ii) the published crystal structure of the CD20 and rituximab complex; and (iii) sequence identity between human and mouse CD20 (BLASTp) based on the assumption that important functional residues would be conserved.

[0175] This approach identified seven (7) serine and threonine residues in the N-terminal cytoplasmic domain of CD20 (“Cl”). These residues are represented in the sequence set forth below in SEQ ID NO: 11 as bold

[0176]

[0177] [SEQ ID NO: 11]

[0178] This approach also identified three (3) serine and threonine residues in the C-terminal cytoplasmic domain of CD20 (“C3”). These residues are also represented in the sequence set forth below in SEQ ID NO: 12 as bold

[0179]

[0180] [SEQ ID NO: 12]

[0181] Accordingly, in another example according to this and other aspects of the application, the at least one mutation associated with feature (iii) comprises a mutation of at least one amino acid residue selected from T2, T3, S7, T11, S35, S36, and T51 set forth in SEQ ID NO: 1.

[0182] In another example according to this aspect of the application, the at least one mutation associated with feature (iv) comprises a mutation of at least one amino acid residue selected from S225, S231, and T239 set forth in SEQ ID NO: 1.

[0183] ​​In yet another example according to this and other aspects of the application, the at least one mutation associated with feature (v) comprises:

[0184] • feature (i) and feature (iv);

[0185] • feature (ii) and feature (iii);

[0186] • feature (i) and a mutation of S225 of SEQ ID NO: 1;

[0187] • feature (i) and a mutation of S231 of SEQ ID NO: 1;

[0188] • feature (i) and a mutation of T239 of SEQ ID NO: 1;

[0189] • feature (i) and a mutation of S225 and S231 of SEQ ID NO: 1;

[0190] • feature (i) and a mutation of S225 and T239 of SEQ ID NO: 1;

[0191] • feature (i) and a mutation of S231 and T239 of SEQ ID NO: 1;

[0192] • feature (i) and a mutation of S225, S231 and T239 of SEQ ID NO: 1;

[0193] • feature (ii) and a mutation of T2 of SEQ ID NO: 1;

[0194] • feature (ii) and a mutation of T3 of SEQ ID NO: 1;

[0195] • feature (ii) and a mutation of S7 of SEQ ID NO: 1;

[0196] • feature (ii) and a mutation of T11 of SEQ ID NO: 1;

[0197] • feature (ii) and a mutation of S35 of SEQ ID NO: 1;

[0198] • feature (ii) and a mutation of S36 of SEQ ID NO: 1;

[0199] • feature (ii) and a mutation of T51 of SEQ ID NO: 1;

[0200] • feature (ii) and a mutation of T2 and T3 of SEQ ID NO: 1;

[0201] • feature (ii) and a mutation of T2 and S7 of SEQ ID NO: 1;

[0202] • feature (ii) and mutations of T2 and T11 of SEQ ID NO: 1 ;

[0203] • feature (ii) and mutations of T2 and S35 of SEQ ID NO: 1 ;

[0204] • feature (ii) and mutations of T2 and S36 of SEQ ID NO: 1 ;

[0205] • feature (ii) and mutations of T2 and T51 of SEQ ID NO: 1 ;

[0206] • feature (ii) and mutations of T3 and S7 of SEQ ID NO: 1 ;

[0207] • feature (ii) and mutations of T3 and T11 of SEQ ID NO: 1 ;

[0208] • feature (ii) and mutations of T3 and S35 of SEQ ID NO: 1 ;

[0209] • feature (ii) and mutations of T3 and S36 of SEQ ID NO: 1 ;

[0210] • feature (ii) and mutations of T3 and T51 of SEQ ID NO: 1 ;

[0211] • feature (ii) and mutations of S7 and T11 of SEQ ID NO: 1 ;

[0212] • feature (ii) and mutations of S7 and S35 of SEQ ID NO: 1 ;

[0213] • feature (ii) and mutations of S7 and S36 of SEQ ID NO: 1 ;

[0214] • feature (ii) and mutations of S7 and T51 of SEQ ID NO: 1 ;

[0215] • feature (ii) and mutations of T11 and S35 of SEQ ID NO: 1 ;

[0216] • feature (ii) and mutations of T11 and S36 of SEQ ID NO: 1 ;

[0217] • feature (ii) and mutations of T11 and T51 of SEQ ID NO: 1 ;

[0218] • feature (ii) and mutations of S35 and S36 of SEQ ID NO: 1 ;

[0219] • feature (ii) and mutations of S35 and T51 of SEQ ID NO: 1 ;

[0220] • feature (ii) and mutations of S36 and T51 of SEQ ID NO: 1 ;

[0221] • feature (ii) and mutations of T2, T3 and S7 of SEQ ID NO: 1 ;

[0222] • feature (ii) and mutations of T2, T3 and T11 of SEQ ID NO: 1 ;

[0223] • feature (ii) and mutations of T2, T3 and S35 of SEQ ID NO: 1 ;

[0224] • feature (ii) and mutations of T2, T3 and S36 of SEQ ID NO: 1 ;

[0225] • feature (ii) and mutations of T2, T3 and T51 of SEQ ID NO: 1 ;

[0226] • feature (ii) and mutations of T2, S7 and T11 of SEQ ID NO: 1 ;

[0227] • feature (ii) and mutations of T2, S7 and S35 of SEQ ID NO: 1 ;

[0228] • feature (ii) and mutations of T2, S7 and S36 of SEQ ID NO: 1 ;

[0229] • feature (ii) and mutations of T2, S7 and T51 of SEQ ID NO: 1 ;

[0230] • feature (ii) and mutations of T2, T11 and S35 of SEQ ID NO: 1 ;

[0231] • feature (ii) and mutations of T2, T11 and S36 of SEQ ID NO: 1 ;

[0232] • feature (ii) and mutations of T2, T11 and T51 of SEQ ID NO: 1 ;

[0233] • feature (ii) and mutations of T2, S35 and S36 of SEQ ID NO: 1 ;

[0234] • feature (ii) and mutations of T2, S35 and T51 of SEQ ID NO: 1 ;

[0235] • feature (ii) and mutations of T2, S36 and T51 of SEQ ID NO: 1 ;

[0236] • feature (ii) and mutations of T3, S7 and T11 of SEQ ID NO: 1 ;

[0237] • feature (ii) and mutations of T3, S7 and S35 of SEQ ID NO: 1 ;

[0238] • feature (ii) and mutations of T3, S7 and S36 of SEQ ID NO: 1 ;

[0239] • feature (ii) and mutations of T3, S7 and T51 of SEQ ID NO: 1 ;

[0240] • feature (ii) and mutations of T3, T11 and S35 of SEQ ID NO: 1 ;

[0241] • feature (ii) and mutations of T3, T11 and S36 of SEQ ID NO: 1 ;

[0242] • feature (ii) and mutations of T3, T11 and T51 of SEQ ID NO: 1 ;

[0243] • feature (ii) and mutations of T3, S35 and S36 of SEQ ID NO: 1 ;

[0244] • feature (ii) and mutations of T3, S35 and T51 of SEQ ID NO: 1 ;

[0245] • feature (ii) and mutations of T3, S36 and T51 of SEQ ID NO: 1 ;

[0246] • feature (ii) and mutations of S7, T11 and S35 of SEQ ID NO: 1 ;

[0247] • feature (ii) and mutations of S7, T11 and S36 of SEQ ID NO: 1 ;

[0248] • feature (ii) and mutations of S7, T11 and T51 of SEQ ID NO: 1 ;

[0249] • feature (ii) and mutations of S7, S35 and S36 of SEQ ID NO: 1 ;

[0250] • feature (ii) and mutations of S7, S35 and T51 of SEQ ID NO: 1 ;

[0251] • feature (ii) and mutations of S7, S36 and T51 of SEQ ID NO: 1 ;

[0252] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0253] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0254] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0255] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0256] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0257] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0258] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0259] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0260] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0261] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0262] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0263] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0264] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0265] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0266] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0267] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0268] • features (i) and (ii) and mutations in T2, S7, T11 and T51 of SEQ ID NO: 1 ;

[0269] • features (i) and (ii) and mutations in T2, S7, S35 and S36 of SEQ ID NO: 1 ;

[0270] • features (i) and (ii) and mutations in T2, S7, S35 and T51 of SEQ ID NO: 1 ;

[0271] • features (i) and (ii) and mutations in T2, S7, S36 and T51 of SEQ ID NO: 1 ;

[0272] • features (i) and (ii) and mutations in T2, T11, S35 and S36 of SEQ ID NO: 1 ;

[0273] • features (i) and (ii) and mutations in T2, T11, S35 and T51 of SEQ ID NO: 1 ;

[0274] • features (i) and (ii) and mutations in T2, T11, S36 and T51 of SEQ ID NO: 1 ;

[0275] • features (i) and (ii) and mutations in T2, S35, S36 and T51 of SEQ ID NO: 1 ;

[0276] • features (i) and (ii) and mutations in T3, S7, T11 and S35 of SEQ ID NO: 1 ;

[0277] • features (i) and (ii) and mutations in T3, S7, T11 and S36 of SEQ ID NO: 1 ;

[0278] • features (i) and (ii) and mutations in T3, S7, T11 and T51 of SEQ ID NO: 1 ;

[0279] • features (i) and (ii) and mutations in T3, S7, S35 and S36 of SEQ ID NO: 1 ;

[0280] • features (i) and (ii) and mutations in T3, S7, S35 and T51 of SEQ ID NO: 1 ;

[0281] • features (i) and (ii) and mutations in T3, S7, S36 and T51 of SEQ ID NO: 1 ;

[0282] • features (i) and (ii) and mutations in T3, T11, S35 and S36 of SEQ ID NO: 1 ;

[0283] • features (i) and (ii) and mutations in T3, T11, S35 and T51 of SEQ ID NO: 1 ;

[0284] • features (ii) and mutations in T3, T11, S36 and T51 of SEQ ID NO: 1;

[0285] • features (ii) and mutations in T3, S35, S36 and T51 of SEQ ID NO: 1;

[0286] • features (ii) and mutations in S7, T11, S35 and S36 of SEQ ID NO: 1;

[0287] • features (ii) and mutations in S7, T11, S35 and T51 of SEQ ID NO: 1;

[0288] • features (ii) and mutations in S7, T11, S36 and T51 of SEQ ID NO: 1;

[0289] • features (ii) and mutations in S7, S35, S36 and T51 of SEQ ID NO: 1;

[0290] • features (ii) and mutations in T11, S35, S36 and T51 of SEQ ID NO: 1;

[0291] • features (ii) and mutations in T2, T3, S7, T11 and S35 of SEQ ID NO: 1;

[0292] • features (ii) and mutations in T2, T3, S7, T11 and S36 of SEQ ID NO: 1;

[0293] • features (ii) and mutations in T2, T3, S7, T11 and T51 of SEQ ID NO: 1;

[0294] • features (ii) and mutations in T2, T3, S7, S35 and S36 of SEQ ID NO: 1;

[0295] • features (ii) and mutations in T2, T3, S7, S35 and T51 of SEQ ID NO: 1;

[0296] • features (ii) and mutations in T2, T3, S7, S36 and T51 of SEQ ID NO: 1;

[0297] • features (ii) and mutations in T2, T3, T11, S35 and S36 of SEQ ID NO: 1;

[0298] • features (ii) and mutations in T2, T3, T11, S35 and T51 of SEQ ID NO: 1;

[0299] • features (i) and (ii) and mutations in T2, T3, T11, S36 and T51 of SEQ ID NO: 1 ;

[0300] • features (i) and (ii) and mutations in T2, T3, S35, S36 and T51 of SEQ ID NO: 1 ;

[0301] • features (i) and (ii) and mutations in T2, S7, T11, S35 and S36 of SEQ ID NO: 1 ;

[0302] • features (i) and (ii) and mutations in T2, S7, T11, S35 and T51 of SEQ ID NO: 1 ;

[0303] • features (i) and (ii) and mutations in T2, S7, T11, S36 and T51 of SEQ ID NO: 1 ;

[0304] • features (i) and (ii) and mutations in T2, S7, S35, S36 and T51 of SEQ ID NO: 1 ;

[0305] • features (i) and (ii) and mutations in T2, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0306] • features (i) and (ii) and mutations in T3, S7, T11, S35 and S36 of SEQ ID NO: 1 ;

[0307] • features (i) and (ii) and mutations in T3, S7, T11, S35 and T51 of SEQ ID NO: 1 ;

[0308] • features (i) and (ii) and mutations in T3, S7, T11, S36 and T51 of SEQ ID NO: 1 ;

[0309] • features (i) and (ii) and mutations in T3, S7, S35, S36 and T51 of SEQ ID NO: 1 ;

[0310] • features (i) and (ii) and mutations in T3, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0311] • features (i) and (ii) and mutations in S7, T11, S35, S36 and T51 of SEQ ID NO: 1 ;

[0312] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35 and S36 of SEQ ID NO: 1 ;

[0313] • features (i) and (ii) and mutations in T2, T3, S7, T11, S35 and T51 of SEQ ID NO: 1 ;

[0314] • features (ii) and mutations of T2, T3, S7, T11, S35, and S36 of SEQ ID NO: 1;

[0315] • features (ii) and mutations of T2, T3, S7, T11, S36, and T51 of SEQ ID NO: 1;

[0316] • features (ii) and mutations of T2, T3, S7, S35, S36, and T51 of SEQ ID NO: 1;

[0317] • features (ii) and mutations of T2, T3, T11, S35, S36, and T51 of SEQ ID NO: 1;

[0318] • features (ii) and mutations of T2, S7, T11, S35, S36, and T51 of SEQ ID NO: 1;

[0319] • features (ii) and mutations of T3, S7, T11, S35, S36, and T51 of SEQ ID NO: 1;

[0320] • features (ii) and mutations of T2, T3, S7, T11, S35, S36, and T51 of SEQ ID NO: 1; and

[0321] • features (iii) and (iv), and including any of the subcombinations just mentioned above.

[0322] In another example according to this and other aspects of the application, the mutation of the at least one serine or threonine located in the amino acid residues 1-56 and 210-297, respectively, shown in SEQ ID NO: 1 comprises a substitution mutation, a deletion mutation, or an insertion mutation.

[0323] In a related example according to this and other aspects of the application, the mutation of the at least one serine or threonine located in the amino acid residues 1-56 and 210-297, respectively, shown in SEQ ID NO: 1 comprises a conservative or non-conservative substitution comprising a naturally or non-naturally occurring amino acid residue.

[0324] Examples of naturally occurring amino acids include, but are not limited to, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Examples of non-naturally occurring amino acids have been reviewed in (47).

[0325] Table 2 in Example 3 summarizes specific examples of modified CD20 proteins, wherein intracellular signaling is reduced or eliminated when expressed by a cell. Sequence specific examples include, but are not limited to, SEQ ID Nos: 2-7, and include combinations of truncations and / or serine / threonine substitution mutations described herein.

[0326] Thus, in yet another aspect, the present application provides a modified CD20 protein comprising or consisting of the sequence set forth in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or a variant sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively.

[0327] Data in Example 3 and Figure 3 indicate that C1C3 (SEQ ID NO: 7) and C1C 252 (SEQ ID NO: 5) mutant proteins attach or bind to the membrane of HEK293 and T cell lines at similar levels observed for the wild-type CD20 protein (SEQ ID NO: 1). Thus, C1C3 (SEQ ID NO: 7) and C1C 252 (SEQ ID NO: 5) mutant proteins were further used for cell signaling assay analysis.

[0328] These data, presented in Examples 7 and 8, further reflect that phosphorylation can play an important role in CD20 + membrane trafficking and intracellular signaling pathways in CD20 KO cell lines. Specifically, in HG3 CD20 KO cell lines, rituximab stimulated C1C3 (SEQ ID NO: 7) or C1C 252(SEQ ID NO: 5) causes impaired signaling, manifested as differential phosphorylation of all proteins measured and Ca 2+ Impaired endocytosis Figure 7 and 8).

[0329] Thus, in another aspect, the present application provides a modified human CD20 protein comprising:

[0330] (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1; and

[0331] (ii) a truncation of amino acid residues 253-297 set forth in SEQ ID NO: 1,

[0332] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0333] In yet another aspect, the present application provides a modified human CD20 protein comprising:

[0334] (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1; and

[0335] (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1

[0336] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0337] wherein the at least one mutation defined by (i) and (ii) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0338] Mutant (human) CD20 - cytoplasmic and extracellular loop domain modifications

[0339] In conjunction with Figure 4 and Figure 5 The preliminary data provided in Examples 4 and 5 indicate that mutation of the predicted phosphorylation sites allows rituximab to induce killing of HG3 cell lines and primary T cells expressing the modified human CD20 proteins described herein. Thus, a transgene incorporating a nucleic acid encoding a modified human CD20 protein described by the present application, for example, is incorporated for expression in a CAR-T cell construct, can be used as a genetic biomarker and / or as a safety / suicide switch to avoid cytotoxicity problems in patients receiving CAR-T cell therapy.

[0340] Accordingly, the modified human CD20 protein according to the application can be further modified in the extracellular loop to induce changes in the epitope recognition sequence targeted by clinically approved monoclonal antibodies, including but not limited to rituximab, obinutuzumab and ocrelizumab, to engineer selective binding (or binding ablation) of these therapeutic molecules. See Example 10.

[0341] Accordingly, in yet another aspect of the application, there is provided a modified human CD20 protein comprising:

[0342] (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 set forth in SEQ ID NO: 1 ;

[0343] (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 set forth in SEQ ID NO: 1 ;

[0344] (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1 ;

[0345] (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1 ;

[0346] (v) a combination comprising any one of (i) to (iv); and

[0347] (vi) a mutation of at least one amino acid residue in amino acid residues 142-188 set forth in SEQ ID NO: 1,

[0348] wherein any one of (i) to (v) results in reduced or ablated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

[0349] In one example according to this aspect of the application, the mutation of at least one amino acid in amino acid residues 142-188 set forth in SEQ ID NO: 1 includes but is not limited to N173, S174 and N176.

[0350] The data provided in Examples 3-5, and the foregoing, in conjunction with the data provided in Figure 7 helps to identify specific constructs that meet this objective (i.e., in addition to mutations in the extracellular loop domain, incorporation of mutations in the cytoplasmic domain defined by C1-C3 and C1-C 252 C2 of the CD20 protein).

[0351] Accordingly, in yet another aspect, the present application provides a modified human CD20 protein comprising:

[0352] (i) a mutation in at least one of T2, T3, S7, T11, S35, S36 and T51 as set forth in SEQ ID NO: 1;

[0353] (ii) a mutation in at least one of N173, S174 and N176 as set forth in SEQ ID NO: 1; and

[0354] (iii) a truncation of amino acid residues 253-297 as set forth in SEQ ID NO: 1.

[0355] In yet another aspect, the present application provides a modified human CD20 protein comprising:

[0356] (i) a mutation in at least one of T2, T3, S7, T11, S35, S36 and T51 as set forth in SEQ ID NO: 1;

[0357] (ii) a mutation in at least one of N173, S174 and N176 as set forth in SEQ ID NO: 1; and

[0358] (iii) a mutation in at least one of S225, S231 and T239 as set forth in SEQ ID NO: 1.

[0359] Expression of mutant (human) CD20

[0360] A modified human CD20 protein according to the present application can be expressed by one or more cells, either in isolation or within a population of cells, for use as, for example, a transgene selection marker comprising the modified human CD20 protein, or as a suicide / safety switch to manage adverse reactions in therapies involving gene transduction or transfection of cells, or as a marker to facilitate detection of the modified cells in a therapeutic recipient.

[0361] Accordingly, in yet another aspect, the present application provides a cell expressing a modified human CD20 protein as described herein, wherein the modified human CD20 protein is trafficked to the cell membrane and attached to or bound to the cell membrane.

[0362] In examples according to this and other aspects of the application, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a B cell, a myeloid cell, a pluripotent stem cell, a non-haematopoietic cell line and a haematopoietic stem cell.

[0363] The present application also contemplates specific cell lines, such as hematopoietic stem cell lines or leukemia-derived cell lines that have been modified to express the human CD20 protein described herein. For example, one method of generating an "off the shelf" CAR therapy involves taking a non-hematopoietic cell line (e.g., K562) and genetically modifying it to reduce alloreactivity and express a CAR as a therapeutic agent.

[0364] Nucleic acids encoding mutant human CD20

[0365] The present application also contemplates nucleic acids (e.g., deoxyribonucleic acid (DNA), messenger ribonucleic acid (mRNA), or complementary deoxyribonucleic acid (cDNA)) encoding the modified human CD20 protein as described herein.

[0366] Thus, in yet another aspect, the present application provides an isolated nucleic acid molecule encoding any of the modified human CD20 proteins as described herein.

[0367] Methods of introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods of introducing polynucleotides (e.g., RNA) into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods, including the Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells by using cationic liposome-mediated transfection, lipofection methods, polymer encapsulation methods, peptide-mediated transfection methods, or biolistic particle delivery systems (e.g., "gene gun") (e.g.,

[24] ).

[0368] Biological methods of introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, such as human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others (e.g., [48, 49]).

[0369] Chemical methods of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle).

[0370] Suitable lipids are available from commercial sources. For example, dimyristyl phosphatidyl choline ("DMPC") is available from Sigma, St. Louis, Mo; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidyl glycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vehicles formed by the deposition of closed bilayered or aggregated lipid structures. Liposomes are characterized by a bilayered membrane structure comprising a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form when excess water is introduced to an appropriate lipid mixture at 60°C. The lipid mixture forms small unilamellar vesicles (SUVs) that are cooled and extruded through a small pore size sieve. The energy of extrusion leads to the production of small uniform SUVs (50-90 nm) that are stable for several hours. The lipid components are self-organized into a bilayer structure prior to the formation of the closed structure, and trap water and dissolved solutes between the lipid bilayers

[50] . However, compositions having structures other than normal vesicular structures in solution are also contemplated. For example, the lipids can assume micellar structures, or exist simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0371] Regardless of the method used to introduce foreign nucleic acids into host cells, a variety of assays can be performed. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of the application.

[0372] In one example, the nucleic acid introduced into the cell (e.g., T cell) is RNA. In another example, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Any source of DNA of interest can be directly converted to a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The template required for in vitro transcription is a modified or chimeric membrane protein, such as a modified human CD20 protein described herein.

[0373] PCR can be used to generate a template for in vitro transcription of mRNA, which is then introduced into a cell. Methods for performing PCR are known in the art. Primers used for PCR are designed to have regions that are substantially complementary to regions of the DNA used as a template for PCR. As used herein, “substantially complementary” refers to nucleotide sequences in which most or all of the bases in the primer sequence are complementary, or one or more bases are not complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to a DNA target of interest under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of a gene that are normally transcribed in a cell (open reading frame), including 5’ and 3’ untranslated regions (UTRs). Primers can also be designed to amplify portions of a gene that encode specific domains of interest. In one example, primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers used for PCR are generated by synthetic methods well known in the art. A “forward primer” is a primer that contains a region of nucleotides that is substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. As used herein, “upstream” refers to a position 5’ of the DNA sequence to be amplified, relative to the coding strand. A “reverse primer” is a primer that contains a region of nucleotides that is substantially complementary to nucleotides of a double-stranded DNA template that are downstream of the DNA sequence to be amplified. As used herein, “downstream” refers to a position 3’ of the DNA sequence to be amplified, relative to the coding strand.

[0374] Chemical structures that can improve RNA stability and / or translation efficiency can also be used. The RNA preferably has 5’ and 3’ UTRs. In one example, the 5’ UTR is between 0 and 3000 nucleotides in length. The length of the 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the length of the 5’ and 3’ UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0375] 5' and 3' UTRs can be the endogenous 5' and 3' UTRs naturally present in the gene of interest. Alternatively, UTR sequences not endogenous to the gene of interest can also be added by incorporating the UTR sequences into the forward and reverse primers, or by any other modification to the template. The use of UTR sequences not endogenous to the gene of interest can be used to alter the stability and / or translation efficiency of the RNA. For example, AU-rich elements in 3' UTR sequences are known to decrease mRNA stability. Thus, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA according to the UTR properties known in the art.

[0376] In one example, the 5' UTR can include a Kozak sequence of the endogenous gene. Alternatively, when adding a 5' UTR not endogenous to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the translation efficiency of certain RNA transcripts, but it does not appear that all RNAs require a Kozak sequence for efficient translation. It is known in the art that many mRNAs require a Kozak sequence. In other examples, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other examples, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0377] To enable the synthesis of RNA from a DNA template without the need for gene cloning, a transcriptional promoter should be linked to the sequence of the DNA template to be transcribed upstream. When the sequence that serves as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one example, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other promoters that can be used include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequence of the T7, T3, and SP6 promoters is known in the art.

[0378] In one example, the 5' end of the mRNA has a cap and the 3' end has a poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability within the cell. On circular DNA templates, such as plasmid DNA, the RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of 3' UTR-terminating linearized plasmid DNA produces mRNA of normal size that cannot be effectively transfected in eukaryotic cells even after post-transcriptional polyadenylation.

[0379] On linear DNA templates, the bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (e.g., [25, 26]).

[0380] A conventional method to integrate a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences integrated into plasmid DNA can cause plasmid instability, and thus plasmid DNA templates obtained from bacterial cells are often severely contaminated with deletions and other aberrations. This makes the cloning process not only laborious and time-consuming, but also often unreliable. Thus, there is an urgent need for a method to construct DNA templates with a polyA / T 3' stretch without the need for cloning.

[0381] A polyA / T stretch of a transcriptional DNA template can be generated during PCR by using a reverse primer containing a polyT tail (e.g., a 100T tail, which can range from 50-5000T in size), or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. A poly(A) tail can also provide stability to RNA and reduce its degradation. Generally, the length of a poly(A) tail is positively correlated with the stability of a transcribed RNA. In one example, the length of a poly(A) tail is between 100 and 5000 adenosines.

[0382] A poly(A) tail of an RNA can be further extended after in vitro transcription using a Poly(A) polymerase (e.g., E. coli Poly(A) polymerase (E-PAP)). In one example, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides can increase the translation efficiency of an RNA by about two-fold. In addition, attaching different chemical groups to the 3' end can improve the stability of an mRNA. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into a poly(A) tail using a Poly(A) polymerase. ATP analogs can further improve the stability of an RNA.

[0383] A 5' cap can also provide stability to an RNA molecule. In one preferred example, an RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (e.g.,

[59] ).

[0384] An RNA produced by the methods disclosed herein can also comprise an internal ribosome entry site (IRES) sequence. An IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to an mRNA and facilitates the initiation of translation. Any suitable solutes for cell electroporation can be included, which can comprise factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0385] In some examples, the RNA encoding the transgene is electroporated into the cell. In one example, the RNA encoding the transgene is in vitro transcribed RNA.

[0386] These methods also enable individualized regulation of expression levels by varying, for example, the promoter or the amount of RNA input, thus enabling regulation of expression levels over a large range. Furthermore, the PCR-based mRNA production technology greatly facilitates the design of mRNAs with different structures and combinations of domains.

[0387] One advantage of the RNA transfection method is that it is essentially transient and vector-free. The RNA transgene can be delivered as a minimal expression cassette to lymphocytes and expressed therein after a brief in vitro cell activation without any additional viral sequences. Under these conditions, the transgene is unlikely to integrate into the host cell genome. Since the RNA transfection is highly efficient and enables uniform modification of the entire lymphocyte population, there is no need to perform cell cloning.

[0388] Gene modification of cells with in vitro transcribed RNA (IVT-RNA) employs two different strategies, both of which have been tested in various animal models in succession. The in vitro transcribed RNA is transfected into the cells by lipofection or electroporation. To enable long-term expression of the transferred IVT-RNA, various modifications are required to stabilize the IVT-RNA.

[0389] Some IVT vectors are known from the literature, which are used as templates for in vitro transcription in a standardized manner and are genetically engineered to produce stable RNA transcripts. The protocols currently used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter for RNA transcription, followed by the gene of interest, which is flanked 3' and / or 5' by untranslated regions (UTRs), and a 3' polyadenyl cassette comprising 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette with a type II restriction endonuclease (the recognition sequence corresponds to the cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this process, some nucleotides remain after linearization as part of the cleavage site and extend or mask the 3' poly(A) sequence.

[0390] RNA gene expression does not require transcription and protein products can be produced rapidly after transfection. Furthermore, since the RNA only needs to reach the cytoplasm and not the nucleus, very high transfection rates can be achieved with typical transfection methods. Furthermore, plasmid-based methods require that the promoter driving expression of the gene of interest remains active in the cell under study.

[0391] In another aspect, the RNA constructs are delivered into cells by electroporation. See, for example, the protocols and methods taught in

[53] for electroporation of nucleic acid constructs into mammalian cells. The various parameters required for electroporation of any known cell type, including electric field strength, are well documented in the relevant research literature as well as in numerous patents and applications in this field

[54] . Equipment for electroporation therapy applications is commercially available, for example, the MedPulser TM DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.) and described in patents such as

[55] ; electroporation can also be used for in vitro cell transfection, as described in

[56] . Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, the administration of nucleic acids comprising expression constructs to cells mediated by electroporation provides an exciting new method for delivering RNAs of interest to target cells using any of the numerous available devices and electroporation systems known to those skilled in the art.

[0392] The application is further described with reference to the following examples. It is to be understood that the claimed application is not in any way limited by these examples.

[0393] Example

[0394] Example 1 : Methodology

[0395] Plasmid construction and bacterial strains

[0396] All gene fragments were computer designed using Benchling (Benchling 2022) and synthesized de novo by IDT (Integrated DNA Technologies, Inc). Fragments for testing in human cell lines or primary cells were cloned into the LeGO plasmid backbone

[27] using Pmel / BamHI restriction enzymes (New England Biolabs) under the EFl promoter. Fragments for testing in mouse cell lines or primary cells were cloned into the pmX_GFP plasmid backbone using Xhol / Pacl enzymes. Plasmids were propagated in chemically competent Stable3 E. coli (F - mcrB mrr hsdS20(r B - ,m B - )recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R xyl-5 λ - leum tl-1) (ThermoFisher Scientific).

[0397] Cell lines and primary cells

[0398] HEK293

[0399] HEK293 cell line for screening CD20 truncations / mutations and lentivirus packaging was obtained from Thermofisher Scientific.

[0400] HG3

[0401] HG3 B cell line CD20 knock-out (CD20KO) was generated by the laboratory of Michal Smida at the Central European Institute for Applied Research and provided along with the CD20+ HG3 control cell line.

[0402] Platinum-E

[0403] Platinum-E cells are a retroviral packaging cell line obtained from Cell Biolabs.

[0404] Human primary T cells

[0405] Primary T cells were isolated from buffy coats obtained from the New Zealand Blood Service (University of Otago Human Ethics Committee (Health) H20-173). Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate columns and Lymphoprep (Stemcell Technologies) and T cells were selected and activated for 24 hours using anti-CD3 / CD28 Dynabeads (ThermoFisher Scientific, 11141D) prior to transduction.

[0406] HEK295FT transfection

[0407] For all experiments screening for CD20 detection on the cell membrane, HEK293 cells were transfected with LeGO_CD20 constructs using lipofectamine3000 (ThermoFisher Scientific, L3000015).

[0408] Lentivirus production

[0409] Lentiviral particles were packaged in HEK293 cells transfected with LeGO_CD20 and third generation lentivirus packaging / structural plasmids pMD2-G, pMDLg and pRSV (Adgene). Lentiviral particles were collected from growth media by centrifugation 48 hours post transfection and frozen at -70°C for later use.

[0410] Human B cell transduction

[0411] Lentiviral particles containing CD20 phosphorylation mutations / truncations were transduced into HG3 cell lines using polybrene (Sigma-Aldrich, TR-1003-G) by spinfection.

[0412] Flow cytometry

[0413] All flow cytometry experiments were performed on a 5 laser (UV-VB-YG-R) Cytek Aurora flow cytometer.

[0414] Detection of surface CD20

[0415] 2H7 conjugated to APC / Fire 750 (Biolegend, 302357), Rituximab conjugated to Alexa Fluor 405 (Novus Biologicals), Obinutuzumab conjugated to APC (Leinco, LT907), or BD Quantibrite TM Surface CD20 was detected with PE mouse anti-human CD20 (L27-PE) (BD Biosciences, 347201).

[0416] In vitro CD20-mediated killing

[0417] CD20KO HG3 cells and CD20WT, C1C3, or C1C 252 CD20KO HG3 cells with constructs were incubated with 10 pg Rituximab (Abeam, ab275973) per mL of serum-free RPMI media in a 96U bottom well plate for 30 minutes at 37°C. Cells were washed to remove unbound Rituximab, then incubated with 25% Baby Rabbit Complement (BioRad, C12CA) diluted with RPMI for 1.5 hours. After this time, cells were stained with Zombie NIR (Biolegend, BIO0423105) and cell viability was assessed by flow cytometry.

[0418] Mouse T cell transduction

[0419] Retroviruses containing pmX_hCD20WT-GFP, pmX_C1C3-GFP, or pmX_C1C 252 -GFP were packaged in Platinum-E cells and used to transduce T cells isolated from C57 mice (B6.SJL-Ptprc a , CD45.2+). Five million transduced cells were transferred intravenously to each mouse (B6.SJL-Ptprc bhCD20WT-GFP, C1C3-GFP, C1C

[0420] In vivo CD20+ T cell depletion

[0421] One day after adoptive transfer, hCD20WT-GFP, C1C3-GFP, C1C 252 -GFP and the mock control group were injected intraperitoneally with 500 ug of 2H7 murine anti-human CD20 antibody (BioXCell, BE0276) and received a second injection of 250 ug of 2H7 the following day. As a control, a group of mice that received hCD20WT-GFP T cells received the same dose of isotype control antibody (BioXCell, BE0086). One day after the second administration, <200 uL of blood was collected from each mouse (animal ethics committee 30148). Lymphocytes were purified and stained with anti-CD45.1-APC Fire750 antibody (Biolegend, 110752), anti-CD19-EF450 antibody (eBioscience, 48-0193-82), anti-TCR beta-PE antibody (BD, 553172) and Zombie NIR antibody (Biolegend, BIO0423105). GFP production was used as a surrogate for hCD20 expression. Cells were analyzed by flow cytometry to assess the percentage of adoptively transferred GFP+ T cells.

[0422] CD20+ cell sorting

[0423] EasySep TM PE positive selection kit to select WT, C1C3 and C1C 252 CD20KO HG3 cells transduced with WT, C1C3 and C1C TM PE murine anti-human CD20. The selection cocktail was added, followed by RapidSpheres TM Incubation of the samples, followed by EasyEights TM magnetic rack (magnet). Selected cells were expanded in RPMI medium (10% FBS and 1% penicillin / streptomycin added, complete RPMI) at 37°C and 5% CO2 and frozen in liquid nitrogen for later use.

[0424] Evaluation of CD20-mediated signaling

[0425] Ca 2+ Intracellular flow assay

[0426] Ca2+ influx assay. Intracellular Ca2+ mobilization induced by rituximab cross-linking was measured by flow cytometry using a calcium influx assay kit (Abeam, 233472). Briefly, 1 million cells were incubated with 10 ug / mL rituximab (RITUXAN®, RIXIMYO, 2583917) for 30 minutes at 37°C. Unbound rituximab was washed away and cells were incubated with 2 mM probenecid (Sigma, P9145) for 10 minutes at 37°C. Cells were then incubated with 2 mM Fluo-4 AM (ThermoFisher, F36206) for 30 minutes at 37°C. Cells were washed and resuspended in ice-cold PBS. Cells were then analyzed by flow cytometry to measure basal Ca2+ flux and peak Ca2+ flux. 2+ Mobilization. Then, the selected WT, C1C3, C1C 252 Transduced and untransduced CD20KO HG3 cells were stained with ZombieNIR and 520 AM Ca 2+ dye for 10 minutes at 37°C. Cells were washed and non-specific antibody binding sites were blocked before incubation with 30 ug / mL rituximab for 30 minutes at 37°C. Unbound rituximab was washed away and cells were incubated with buffer for 45 minutes at 37°C. Basal Ca2+ flux was measured and 200 ug / mL goat anti-human IgG hypercross-linking antibody (Invitrogen, 62-8400) was added to measure peak Ca2+ flux. 2+ fluorescence intensity and 200 ug / mL goat anti-human IgG hypercross-linking antibody (Invitrogen, 62-8400) was added to measure peak Ca2+ flux. 2+ fluorescence intensity.

[0427] CD20-mediated phosphorylation

[0428] One million transduced CD20 mutant HG3 CD20KO cells were stimulated with 10 ug / mL rituximab (RITUXAN®, RIXIMYO, 2583917) for 24 hours. CD20-mediated phosphorylation was assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D systems, ARY003C) and pixel intensity for each array spot was analyzed using Image Lab software according to the manufacturer’s instructions.

[0429] Statistical analysis

[0430] Data analysis was performed using GraphPad Prism 10 software. Values are expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed using two-way ANOVA with multiple comparisons, with p<0.05 considered significant.

[0431] Example 2: No truncated / chimeric CD20 detected on cell membrane

[0432] The initial strategy to design modified CD20 molecules with abrogated intracellular signaling was: (i) truncating most of the cytoplasmic domain and (ii) combining the cytoplasmic and transmembrane domains of CD20. Table 1 lists the various sequence constructs developed for these initial experiments, including CD20t, CD20raft, CD20loop, CD20raft v2, CD20loop v2, CD20t v3.1, and CD20t v3.2.

[0433] Since significant modifications to the transmembrane 4a (MS4a) protein can affect its structure and its ability to be transported to and properly attached to or bound to the cell membrane, the applicant sought to investigate the effects of truncating different CD20 domains.

[0434] Table 1. Summary of CD20 truncated / chimeric constructs not detected on the cell membrane

[0435]

[0436]

[0437] The applicant also combined the minimal antibody-binding epitope of CD20 with different signal peptides, transmembrane domains, and cytoplasmic domains known to promote good surface protein transport and expression

[15] . The applicant first tested the human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) signal peptide and the epidermal growth factor receptor (EGFR) transmembrane domain because truncating the intracellular domain of EGFR produces membrane-binding proteins

[15] . See the constructs designated as CD20vt v4 and CD20t v5.1.

[0438] Next, the applicant combined antibody-binding regions of CD20 of varying lengths with the CD28 signal peptide and with transmembrane and cytoplasmic domains derived from CD28 and contactin-associated protein-like 2 (CASPR2) to generate chimeric proteins, as constructs incorporating the transmembrane / cytoplasmic domains of CD28 and CASPR2 can form membrane-bound proteins [16,17]. See constructs designated as CD20t v5.2, CD20t v5.3, and CD20t v5.4.

[0439] refer to Figure 1 The data provided indicate that although the CD20 gene is expressed (as reflected by the production of green fluorescent protein), no truncated or chimeric CD20 molecules were detected on the HEK293 cell membrane.

[0440] Example 3: Phosphorylation-weakened CD20 detected on cell membrane

[0441] Since no truncated or chimeric CD20 was detected on the HEK293 cell membrane, the applicant changed the method to prepare a membrane-bound non-signal transduction CD20 molecule.

[0442] In normal and malignant B cells, serine and threonine residues of CD20 are highly phosphorylated, a process that is associated with B cell proliferation

[18] . Binding of rituximab to CD20 initiates a cascade of signals that can play a role in antibody-mediated cell killing. CD20 associates with lyn, fyn, lck and p75 / 85 kinases

[19] , which activate PLCy through src family kinases

[20] .

[0443] Although reports on the function of CD20 contradict each other, phosphorylation and / or association with kinases appear to be important mechanisms of its activation, and therefore, a CD20 molecule mutated at the phosphorylation sites would retain the antibody binding region but would be unable to transduce signals.

[0444] Most phosphorylation events in eukaryotic cells occur on serine (S), threonine (T) and tyrosine (Y) residues

[28] . The cytoplasmic sequence of CD20 contains 15 serine, 11 threonine and no tyrosine residues ( Figure 2 ). Therefore, CD20 has 26 possible phosphorylation sites, but there is currently direct evidence for only two sites

[21] . To reduce the number of introduced modifications and thus the alterations in protein structure, the applicants used three different predictors to select potential phosphorylation modification sites (i) NetPhos 3.1, which uses a set of neural networks to predict S, T or Y phosphorylation sites in eukaryotic proteins; (ii) the published crystal structure of the CD20 and rituximab complex; and (iii) sequence identity between human and mouse CD20 (BLASTp) based on the assumption that important functional residues would be conserved.

[0445] Combining these approaches, putative phosphorylation sites in cytoplasmic regions 1 and 3 were identified ( Figure 2 ).

[0446] Ten amino acids that were phosphorylated in the crystal structure and / or predicted by NetPhos and identified by the mouse sequence were substituted with alanine (A) because it eliminates the amino acid side chain but does not alter the main chain conformation nor create major electrostatic or steric changes

[29] .

[0447] Julie Deans et al. designed CD20 variants containing these phosphorylation mutations and conserved truncations for studying their intermolecular interactions with CD20

[19] . See Table 2 below:

[0448] Table 2. Combinations of CD20 targeted mutations and truncations

[0449]

[0450]

[0451] Mutant / truncated versions of the CD20 gene contained in the LeGO plasmid backbone were transfected into HEK293 cells. All combinations of phosphorylation mutations and conserved truncations of the CD20 gene were detected on the HEK293 cell membrane with varying expression levels. Next, the applicants used lentivirus containing the phosphorylation mutated / truncated CD20 transgene to transduce primary T cells Figure 3

[0452] While all truncated / mutated CD20 molecules were detected on the HEK293 cell membrane Figure 3 ), the C 252 (SEQ ID NO: 9) and N 51 (SEQ ID NO: 8) proteins were not detected on the primary T cell surface and truncation of both cytoplasmic chains resulted in lower expression levels of C 51 C 252 (SEQ ID NO: 10). Surprisingly, when the individual truncations were combined with the mutations of C1 and C3, respectively, membrane detection was restored Figure 3 ).

[0453] Since cells producing the C1C3 (SEQ ID NO: 7) and C1-C 252 proteins had similar membrane levels as CD20WT, further studies focused on evaluating these two CD20 variants.

[0454] Example 4: Mutations at predicted phosphorylation sites allow rituximab to mediate CDC in vitro in CD20 mutant-transduced HG3 cells Figure 4A

[0455] CD20KO HG3 cells transduced with LeGO_CD20WT, LeGO_C1C3 or LeGO_C1-C 252 and a mock transduced control were incubated with rituximab and CDC was induced using baby rabbit complement. Rituximab mediated significant cytotoxicity against CD20 Figure 4B and was not affected by the predicted phosphorylation site mutations or truncation of the cytoplasmic domain 3. Differences in transduction efficiency of the individual mutants and CD20WT Figure 6 led to differences in cytotoxicity and the observed <100% cytotoxicity.

[0456] Example 5: In vivo specific depletion of hCD20WT+, C1C3 and C1C 252 T cells

[0457] hCD20WT-GFP, C1C3-GFP, C1C 252 ​-GFP mouse T cells were adoptively transferred into mice. Following administration of the anti-CD20 antibody 2H7, a rituximab surrogate suitable for mice, the hCD20+ cell population was significantly reduced (~65%) in all mice compared to mice receiving control antibody. Importantly, the depleted fraction was composed of the highest transgene-expressing cells, and C1C3-GFP and C1C 252 -GFP cells were reduced equally, indicating that intracellular mutation and / or truncation of CD20 did not affect 2H7-mediated killing.

[0458] Example 6: Various anti-CD20 antibodies can detect C1C3 and C1C 252

[0459] The FDA recommends that long-term follow-up studies of integrating vectors should last 15 years

[30] . Applicants propose that their novel CD20 proteins can be used not only as a safety switch for cell therapy, but also as a selectable marker for long-term detection of genetically modified cells. Applicants tested three anti-CD20 antibodies for binding to CD20WT, C1C3, and C1C 252 Binding of sorted CD20+ transduced HG3 cells by Figure 7 Ocrelizumab-APC, Rituximab-AF405, and L27-PE all detect CD20WT and mutants. More importantly, L27-PE, which is clinically relevant and used in hospital clinical laboratories to detect CD20+ cells, can also be used to detect cell therapies including mutant CD20.

[0460] Example 7: Mutation of phosphorylation site attenuates CD20 ligation-induced Ca2+ influx 2+ Inflow

[0461] CD20 cross-linking, or cross-linking of CD20-bound rituximab, can stimulate CD20 mobilization to lipid rafts and increase Ca 2+ influx

[31] . In many cell types, Ca 2+ influx promotes cell activation, proliferation, and / or differentiation

[32] , and Ca 2+ influx can not be ideal when trying to deplete genetically modified cell populations. Cross-linking of CD20WT, C1C3, and C1C 252 or Ca 2+ influx induced in the absence of CD20 was evaluated in the CD20KO HG3 B cell line Example 8: B cell phosphorylation status is affected by C1C3 activation ). CD20 mutant-induced Ca 2+ influx levels were significantly lower than CD20WT (P < 0.01) and comparable to levels in untransduced CD20KO cells, indicating that mutation of intracellular CD20 phosphorylation sites can prevent Ca 2+ influx and its downstream effects on CD20 cross-linking.

[0462] Example 9: Therapeutic utility of modified CD20 proteins

[0463] Phosphokinase activity is a major functional readout of signaling proteins. Akt, p38MAPK, and ERK are common kinases that play a key role in signaling pathways involved in T cell and B cell activation [33, 34]. CD20 is a key signaling protein that regulates B cell activation

[35] , and is phosphorylated to different extents in resting and activated cells. We evaluated the effect of mutations in predicted phosphorylation sites of CD20 on the global phosphorylation state of B cell downstream kinases upon CD20 engagement. C1C3 activation induced by rituximab resulted in a phosphorylation state comparable to CD20-deficient B cells (Figure 8), suggesting that CD20 regulation of B cell activation can be reduced by impairing CD20 phosphorylation. Phosphorylation of cytoplasmic domain 3 appears to be highly relevant, as despite mutations in phosphorylation sites in cytoplasmic domain 1, C1C3 activation resulted in a phosphorylation state comparable to CD20 WT. Notably, phosphorylation of ERK1 / 2 activates transcription factors involved in B cell and T cell proliferation and survival. While C1C3 activation induced phosphorylation of these kinases comparable to CD20 WT, C1C3 activation resulted in a ~3-fold reduction in phosphorylation of ERK1 / 2, which would not only result in reduced activation of B cells, but also reduced activation of T cells. 252 The phosphorylation pattern shown is comparable to CD20 WT. Notably, phosphorylation of ERK1 / 2 activates transcription factors involved in B cell and T cell proliferation and survival. While C1C3 activation induced phosphorylation of these kinases comparable to CD20 WT, C1C3 activation resulted in a ~3-fold reduction in phosphorylation of ERK1 / 2, which would not only result in reduced activation of B cells, but also reduced activation of T cells. 252 The phosphorylation pattern shown is comparable to CD20 WT. Notably, phosphorylation of ERK1 / 2 activates transcription factors involved in B cell and T cell proliferation and survival. While C1C3 activation induced phosphorylation of these kinases comparable to CD20 WT, C1C3 activation resulted in a ~3-fold reduction in phosphorylation of ERK1 / 2, which would not only result in reduced activation of B cells, but also reduced activation of T cells.

[0464] Example 10: Extracellular mutations exclusive of rituximab or obinutuzumab binding

[0465] The transgenes encoding the modified CD20 proteins according to the present application can potentially be incorporated into the following genetically modified cells: (a) T cells, including CAR-T cells

[36] , T cells expressing transgenic T cell receptors (TCRs)

[37] , or T cells expressing other transgenic or synthetic proteins

[38] ; (b) natural killer (NK) cells

[39] ; (c) B cells

[40] ; (d) myeloid cells

[41] , including myeloid cells modified to express transgenic receptors (e.g., CARs, TCRs, B cell receptors (BCRs), or NK receptors) or to express other transgenic or synthetic proteins; (e) cell lines, including induced pluripotent stem cell (iPSC)-derived cell lines

[42] and hematopoietic-derived cell lines, wherein the cell lines have been modified to express transgenic or synthetic proteins; (f) hematopoietic stem cells

[43] modified to express transgenic or synthetic proteins, or edited to correct genetic defects, wherein the transgenic or synthetic proteins have therapeutic benefit.

[0466] Furthermore, nucleic acid molecules encoding the modified CD20 proteins described herein can be comprised in RNA or DNA products aimed at eliciting transient transfection or long-term transduction of stem cells, non-hematopoietic stem cells and immune cells, or in viral vectors aimed at eliciting transient or long-term expression of transgenes, both in vivo in human recipients.

[0467] In each case, the modified CD20 can be used as: (i) a tag to identify, select or purify genetically transduced cells during and after the cell product manufacturing process; (ii) a tag to identify and / or characterize genetically transduced or transfected cells in patient-derived samples or biopsy samples, including by flow cytometry or immunohistochemistry methods; and / or (iii) as a safety switch to rapidly deplete genetically transduced cells using a therapy against CD20 (e.g. rituximab, obinutuzumab or ocrelizumab).

[0468] The modified CD20 proteins incorporating only intracellular phosphorylation site changes retain the native transmembrane and extracellular CD20 sequence. This reduces the risk of antibody-mediated immunogenicity against genetically transduced or transfected cells compared to modified CD20 proteins incorporating multiple extracellular mutations.

[0469] Figure 9A

[0470] Circulating levels of rituximab or obinutuzumab can limit the use of CD20 as a safety switch in patients with B-cell malignancies who received treatment with these antibodies prior to gene therapy. In the present application, the applicants mutate the second extracellular loop of CD20 to allow exclusive binding of rituximab or obinutuzumab.

[0471] In 2013, Klein et al. reviewed the epitope interactions of monoclonal antibodies targeting CD20 [8] (Figure 9).

[0472] Asparagine 171 is an amino acid essential for the binding of rituximab to CD20, however, the binding of obinutuzumab is maintained when this amino acid is substituted by almost any other amino acid. Mutation of N176 abrogates the binding of obinutuzumab, but maintains the binding of rituximab. Conservative substitution of these asparagine residues to aspartic acid (i.e. underlined In Example 11 : ConclusionN176D), CD20 forms that exclusively bind obinutuzumab or rituximab, respectively, can be generated. Applicants designed a Delta obinutuzumab mutant (CD20 N176D) and a Delta rituximab mutant (CD20 N171D). The Delta obinutuzumab mutant retains binding of rituximab and obinutuzumab, however, due to the widespread use of rituximab, the Delta rituximab mutant does not allow rituximab binding but retains obinutuzumab binding. CD20 molecules that combine this extracellular point mutation with an intracellular mutation (C1C3) can be used for patients that have previously received rituximab therapy and are prescribed a CAR-T cell therapy with a safety switch. Detection of anti-CD20 antibodies as well as CD20-mediated killing of this protein (Delta rituximab-C1C3) will be tested in further studies.

[0473] ​

[0474] The tertiary structure of CD20 cannot be maintained by major disruption of its secondary structure, however, minor changes can successfully produce a protein that can be detected at the cell surface. Applicants designed putative CD20 phosphorylation mutants that can be detected in the cell membrane of HEK293 (human embryonic kidney) cell lines, HG3 (B cells), and most importantly, primary T cells. These phosphorylation mutants impair the CD20 signaling cascade in B cells, such as Ca 2+ influx and global kinase phosphorylation, while allowing CD20 antibody detection, and antibody-dependent cellular cytotoxicity in B cells in vitro and T cells in vivo. By mutating key residues in the predicted phosphorylation sites of CD20, Applicants were able to retain cell surface expression, detection, and cell death induction by clinically relevant antibodies, while eliminating the signaling function of CD20. This maximizes the potential risk of cell engagement by safety switch engagement. Although both CD20 mutants eliminate Ca 2+ influx, only C1C3 has an impact on the phosphorylation status of B cells, highlighting the relevance of the phosphorylation sites in cytoplasmic domain 3.

[0475] Accordingly, the CD20 phosphorylation mutants described herein are strong candidates for developing a safety switch and detection tool or as a selection marker for genetically modified cell therapies.

[0476] ***

[0477] While the application has been described by example, it will be appreciated that modifications and variations of the application can be made by those skilled in the art in light of the foregoing teachings. Also, it will be appreciated that where specific features are mentioned, these equivalents will be incorporated as if specifically mentioned in the specification.

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Claims

1. A human CD20 protein comprising: (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 set forth in SEQ ID NO: 1; (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 set forth in SEQ ID NO: 1; (iii) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1; (iv) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO: 1; or (v) a combination comprising any one of (i) to (iv) wherein any one of (i) to (v) results in reduced or eliminated intracellular signaling when the modified CD20 protein is attached to or bound by a cell membrane.

2. The human CD20 protein of claim 1, comprising: (i) at least one mutation that results in truncation of any one or more of amino acid residues 1-56 set forth in SEQ ID NO: 1; and (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO:

1.

3. The human CD20 protein of claim 1, comprising: (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1; and (ii) at least one mutation that results in truncation of any one or more of amino acid residues 210-297 set forth in SEQ ID NO:

1.

4. The human CD20 protein of claim 1, comprising: (i) at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1; and (ii) at least one mutation of a threonine or serine located in amino acid residues 210-297 set forth in SEQ ID NO:

1.

5. The human CD20 protein of claim 1 or claim 2, wherein amino acid residues 1-50 set forth in SEQ ID NO: 1 are truncated.

6. The human CD20 protein of claim 1 or claim 3, wherein amino acid residues 253-297 set forth in SEQ ID NO: 1 are truncated.

7. The human CD20 protein of claim 1, claim 3, or claim 4, wherein the at least one mutation of a threonine or serine located in amino acid residues 1-56 set forth in SEQ ID NO: 1 comprises a mutation of any one or more of T2, T3, S7, T11, S35, S36, and T51.

8. The human CD20 protein of claim 1, claim 2 or claim 4, wherein the mutation of at least one threonine or serine located in amino acid residues 210-297 of SEQ ID NO: 1 comprises a mutation of any one or more of S225, S231 and T239.

9. The human CD20 protein of any one of claims 1 to 8, wherein the at least one mutation is selected from the group consisting of a substitution mutation, a deletion mutation and an insertion mutation.

10. The human CD20 protein of claim 9, wherein the substitution mutation comprises a substitution with at least one naturally or non-naturally occurring amino acid residue.

11. The human CD20 protein of claim 7, wherein the mutation is selected from any one or more of T2A, T3A, S7A, T11A, S35A, S36A and T51A.

12. The human CD20 protein of claim 8, wherein the mutation is selected from any one or more of S225A, S231A and T239A.

13. The human CD20 protein of claim 1, comprising or consisting of the sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, respectively.

14. The human CD20 protein of any one of claims 1 to 13, further comprising at least one mutation of the extracellular domain defined by amino acid residues 142-188, preferably amino acid residues 167-183, of SEQ ID NO: 1, which mutation abrogates binding of rituximab or obinutuzumab or ocrelizumab.

15. The human CD20 protein of claim 14, wherein the at least one mutation comprises a mutation of amino acid residues N171, S173 and / or N176 of SEQ ID NO:

1.

16. A cell expressing the human CD20 protein of any one of claims 1 to 15.

17. The cell of claim 16, comprising a T cell, a natural killer cell, a B cell, a myeloid cell, a pluripotent stem cell, a hematopoietic stem cell, a non-hematopoietic stem cell and a human cell.

18. A nucleic acid comprising a sequence encoding the human CD20 protein of any one of claims 1 to 15.

19. A vector comprising the nucleic acid of claim 18.

20. The vector of claim 19, which is a viral vector.

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